CN110407943B

CN110407943B - Nano antibody of carbofuran pesticide and preparation method and application thereof

Info

Publication number: CN110407943B
Application number: CN201910341232.6A
Authority: CN
Inventors: 王弘; 张瑾如; 杨金易; 徐振林; 孙远明; 沈玉栋; 肖治理
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-04-25
Filing date: 2019-04-25
Publication date: 2021-03-30
Anticipated expiration: 2039-04-25
Also published as: WO2020215727A1; CN110407943A

Abstract

The invention discloses a nano antibody of carbofuran pesticide and a preparation method and application thereof, wherein the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1; the nucleotide sequence of the gene for coding the gene is shown as SEQ ID NO. 2. The nano antibody for the carbofuran pesticide can detect the carbofuran pesticide, has accurate detection result, good effect and good stability, and has good detection effect under the conditions of high temperature and organic solvent. The antibody can be widely applied to detection of carbofuran pesticide residue in agricultural products; the mutant can be used as a precursor, is modified by random or site-directed mutagenesis technology, can obtain mutants with better properties (affinity, specificity, stability and the like), is used for further development and application in the fields of food, medicine, agriculture and the like, and has great application and popularization values.

Description

Nano antibody of carbofuran pesticide and preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, and particularly relates to a nano antibody of carbofuran pesticide, and a preparation method and application thereof.

Background

The carbamate compounds are used as insecticides, acaricides, herbicides and bactericides on pesticides, and form a large class of pesticides with multiple varieties, good pesticide effect and low toxicity. Carbamate pesticides are the main cause of acute pesticide poisoning and are key detection varieties of pesticide residues in vegetables.

Carbofuran, a trade name, is a carbamate broad-spectrum systemic insecticide, acaricide, nematicide, and can be used for controlling more than 300 kinds of pests and nematodes in soil and on the ground for various crops. And has the functions of shortening the growth period of crops and promoting the growth and development of the crops so as to effectively improve the yield of the crops. Is widely applied to pest control of vegetables, fruits, grain crops and the like. However, carbofuran has high toxicity to human and animals, is not easy to degrade, and easily causes environmental pollution. China has stipulated the maximum residue limit of carbofuran in food, but the phenomenon of illegal use is still more, so that the strengthening of the detection of carbofuran pesticide residue is necessary.

At present, the carbofuran residue analysis generally uses gas chromatography (GC for short), high performance liquid chromatography (HPLC for short) and chromatography-mass spectrometry combined technology, the methods are sensitive and accurate, and can simultaneously measure a plurality of medicines, but the sample pretreatment is complex, tedious and time-consuming, expensive instruments and equipment and professional operators are needed, the detection cost is high, and the requirements of on-site, batch and rapid detection of the sample are difficult to meet. Therefore, it is of great significance to develop a simple and rapid analysis method suitable for on-site monitoring of pesticide residues. The immunoassay method established based on the antibody has the advantages of rapidness, sensitivity and high flux, but the antibody is often poor in stability and easy to inactivate under extreme conditions.

Therefore, a carbofuran antibody with good stability is lacked at present.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a nano antibody of carbofuran pesticide and a preparation method and application thereof.

The first purpose of the invention is to provide a nano antibody which can specifically recognize carbofuran.

The second purpose of the invention is to provide a gene for coding the carbofuran nano antibody which can specifically recognize carbofuran.

The third object of the present invention is to provide a recombinant vector.

It is a fourth object of the present invention to provide a recombinant cell.

The fifth purpose of the invention is to provide the application of one or more of the nano antibody, the gene, the recombinant vector or the recombinant cell in the detection of carbofuran or the preparation of a detection kit of carbofuran.

The sixth purpose of the invention is to provide a preparation method of the nano antibody.

A seventh object of the present invention is to provide a method for detecting carbofuran.

An eighth object of the present invention is to provide a kit for detecting carbofuran.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the nano antibody has good stability and higher affinity, overcomes the defects of small molecule functional antibodies, and has the advantages of small molecular mass, weak immunogenicity, strong tissue penetrating power and the like which are not possessed by monoclonal antibodies and polyclonal antibodies. The invention screens out nano antibodies capable of being specifically combined with target molecules (carbofuran complete antigen) from a camel source immune single-domain heavy-chain antibody library by a phage display technology, and establishes a method for quickly, sensitively and stably detecting carbofuran residues in vegetables and tea.

The invention therefore claims the following:

a nano antibody for specifically recognizing carbofuran has an amino acid sequence shown in SEQ ID NO. 1.

The nucleotide sequence of the gene for coding the specificity recognition carbofuran nano antibody is shown as SEQ ID NO. 2.

A recombinant vector which is connected with the gene.

Preferably, the vector is an expression vector.

Preferably, the expression vector is pComb3 xss.

A recombinant cell, a cell carrying the recombinant vector, or a cell capable of expressing the nanobody.

Preferably, the cell is E.coli BL21(DE 3).

The application of one or more of the nano antibody, the gene, the recombinant vector or the recombinant cell in the detection of carbofuran or the preparation of a detection kit of carbofuran.

The preparation method of the nano antibody comprises the following steps:

s1, connecting the coding gene of the nano antibody to an expression vector to obtain a recombinant vector;

s2, transferring the recombinant vector into a receptor cell to obtain a recombinant cell;

s3, culturing the recombinant cells, and performing induced expression on the nano antibody;

and S4, separating and purifying to obtain the nano antibody.

Preferably, in step S3, the recombinant cell is cultured until the log-phase OD600 value is 0.6-0.8, and the nanobody is induced to be expressed.

Preferably, in step S3, 0.1-1 mM IPTG is used for inducing expression for 12-16 h at 28-37 ℃.

More preferably, in step S3, expression is induced for 12h at 37 ℃ using 1mM IPTG.

Preferably, in step S4, periplasmic cavity proteins are extracted by sucrose osmotic pressure method, and soluble nanobodies in the periplasmic cavity are recovered after one-step Ni column purification.

A carbofuran detection method, which utilizes the nano-antibody.

Preferably, the detection is carried out based on an indirect ELISA method, carbofuran complete antigen obtained by coupling carbofuran hapten and carrier protein in the formula (I) is used as a coating antigen, the nano antibody is used as a detection antibody,

preferably, the carrier protein is ovalbumin.

More preferably, the detection method comprises the steps of: coating the enzyme-labeled plate with the coating antigen; adding a carbofuran standard substance or a sample to be detected, and then adding a detection antibody; adding enzyme-labeled secondary antibody, and incubating; adding a color development solution, and incubating; adding a stop solution and measuring; and establishing a standard curve by taking the log10 value of the concentration of the carbofuran standard substance as an abscissa and taking the ratio of the light absorption value of each carbofuran standard substance concentration to the light absorption value of the zero standard hole as an ordinate, and further calculating the content of the carbofuran in the sample to be detected according to the light absorption value of the sample to be detected.

More preferably, the carbofuran hapten and the egg white albumin are coupled to obtain the carbofuran complete antigen serving as a coating antigen, the coating antigen is diluted to the working concentration of 1 mug/mL, an enzyme label plate is added, each hole is 100 mug L, and the incubation is carried out overnight at 37 ℃; washing the plate for 2 times by a plate washing machine, drying liquid in the holes by spinning, adding 120 mu L of confining liquid, incubating for 2h at 37 ℃, drying the liquid in the holes by spinning, inverting, and drying for 1h at 37 ℃; adding 50 mu L of carbofuran standard substance or sample to be detected into each hole, adding 50 mu L of the nano antibody, incubating for 1h at 37 ℃, washing the plate for 5 times by a plate washing machine, and spin-drying liquid in the holes; adding 100 μ L anti-HA-HRP antibody (20000 times diluted) into each well, incubating at 37 deg.C for 40min, washing the plate for 5 times, and spin-drying the liquid in the wells; adding 100 μ L of TMB color development solution into each well, and incubating at 37 deg.C for 10 min; then 50. mu.L of 10% H was added₂SO₄Terminating the color development reaction; measuring the light absorption value of A450nm by an enzyme-labeling instrument; the standard curve was fitted using a four parameter fitting model from origin9.0 with the abscissa being the log10 value for the concentration of carbofuran drug and the ordinate being the absorbance ratio B/B0.

A kit for detecting carbofuran, which contains the nano-antibody.

Preferably, the detection is carried out based on an indirect ELISA method, and the detection also comprises carbofuran complete antigen obtained by coupling carbofuran hapten and carrier protein as a coating antigen, the nano antibody is used as a detection antibody,

preferably, the carrier protein is ovalbumin.

Preferably, the kit also comprises an enzyme-labeled secondary antibody, a developing solution and a stop solution.

Compared with the prior art, the invention has the following beneficial effects:

the nano antibody for the carbofuran pesticide can detect the carbofuran pesticide, has accurate detection result, good effect and good stability, and has good detection effect under the conditions of high temperature and organic solvent. The antibody can be widely applied to detection of carbofuran pesticide residue in agricultural products; the mutant can be used as a precursor, is modified by random or site-directed mutagenesis technology, can obtain mutants with better properties (affinity, specificity, stability and the like), is used for further development and application in the fields of food, medicine, agriculture and the like, and has great application and popularization values.

Drawings

FIG. 1 shows the detection result of indirect competitive ELISA of phage monoclonal.

FIG. 2 is a standard curve of detecting carbofuran pesticide by nano antibody.

FIG. 3 shows the binding capacity of the nanobody at high temperature for 5 min.

FIG. 4 shows the binding activity of nanobodies at 95 ℃.

FIG. 5 shows the binding capacity of antibodies to antigens using methanol as a diluent.

FIG. 6 shows the binding capacity of an antibody to an antigen in acetonitrile as a diluent.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 construction of immune library of nano antibody against carbofuran pesticide

First, experiment method

1. Preparation of complete antigens BFNB-OVA and BFNB-KLH

The complete antigens BFNB-OVA and BFNB-KLH are prepared by coupling the carbofuran pesticide hapten BFNB with ovalbumin OVA (albumin) and keyhole limpet hemocyanin KLH (keyhole limpet hemocyanin).

The chemical formula of the carbofuran pesticide hapten BFNB is as follows:

2. immune Bactrian camel

500 μ g of BFNB-KLH was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into the neck of a bactrian camel at multiple points. The boosting immunization is carried out once every 2 weeks, 500 mu g of BFNB-KLH and Freund's incomplete adjuvant in equal volume are emulsified and then immunized, and blood is collected intravenously after one week of each immunization. The serum titer was determined by indirect competition ELISA.

3. Extraction of RNA and cDNA Synthesis

Blood samples with the best serum inhibition were taken for lymphocyte isolation and RNA extraction. The extraction of RNA was performed according to the Trizol reagent method of Invitrogen corporation. First strand cDNA synthesis was performed using RNA as a template, according to the TAKARA first strand reverse transcription kit instructions.

4. Obtaining of Nano antibody Gene fragment

The variable region coding gene of the camel heavy chain antibody is obtained by PCR amplification by using Taq Mix DNA polymerase (the primers are shown in the following table 1).

Table 1 primer sequences for amplification of VHH genes:

respectively carrying out one-step PCR and two-step PCR on the first strand of the cDNA obtained in the last step

(1) The one-step PCR adopts primers P1 and P2 and primers P1 and P3 to respectively amplify target fragments under the reaction conditions of 94 ℃, 5min, 94 ℃, 30s, 61 ℃, 30s, 72 ℃, 1min, 30 cycles and extension at 72 ℃ for 10 min. Respectively carrying out agarose gel electrophoresis on the PCR products, recovering fragments of about 500bp, recovering target fragments through a DNA gel cutting recovery kit, combining to obtain a nano antibody gene fragment, quantifying, and storing at-20 ℃ for later use;

(2) two-step PCR amplification the first step PCR was amplified with primers Q1 and Q2 under the conditions of 94 ℃, 5min, 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min, 30 cycles, and extension at 72 ℃ for 10 min.

And (3) carrying out agarose gel electrophoresis on the PCR product of the first step, recovering fragments of about 500bp, and recovering target fragments by using a DNA gel cutting recovery kit. The recovered target fragment is used as a template, and the second step of amplification is carried out by using primers Q3 and Q4, and the PCR reaction conditions are the same as the first step. And further cutting and recovering the gel to obtain a nano antibody gene fragment, quantifying, and storing at-20 ℃ for later use.

5. Preparation of nano antibody gene library

And carrying out sfiI double enzyme digestion on the phagemid vector pComb3xss and the two nano antibody gene fragments obtained in the previous step, and obtaining the pComb3xss and the two nano antibody gene fragments through gel cutting recovery and PCR purification recovery. Then at 16 ℃ the mixture was purified with pComb3xss and fragment of interest 1: 3 and the reaction was performed overnight with T4 ligase.

The ligation product was recovered by precipitation with a PCR purification kit and dissolved in 35. mu.l of sterile water. The one-step and two-step processes were connected to product 1: 1, mixing, electrically transforming into competent cells ER2738 in 13 times, and culturing the transformed bacteria liquid in a shaking table at 200rpm and 37 ℃ for 1 hour to recover the growth. The transformed bacteria were diluted in gradient and plated on LB plates containing ampicillin and tetracycline, and cultured overnight at 37 ℃. The next day, plate-coated monoclonal antibodies were randomly picked and sent to the company for sequencing to identify the diversity of the antibody library. The library capacity was calculated based on the number and diversity of clones.

And scraping and washing the cells by using a culture medium, adding sterile glycerol with the final concentration of 20 percent, and preserving at the temperature of minus 80 ℃ to obtain the carbofuran pesticide nano antibody gene bank.

6. Preparation of Nanobody phage library

The frozen nanobody pool (1 mL) was inoculated into 200mL LB (Amp, Tet) medium, and cultured at 37 ℃ and 250rpm with shaking until logarithmic phase (OD 600. RTM. 0.6-0.8). 1mL of helper phage (1X 1012pfu/mL) was added, incubated at 37 ℃ for 30min with shaking at 250rpm for 2h, and then kanamycin (helper phase resistance) was added to 70. mu.g/mL, and incubated overnight with shaking at 250 rpm. The culture broth was centrifuged at 12000rpm for 20min at 4 ℃ and the supernatant was transferred to a new centrifuge flask, 50mL of 5xPEG/NaCl was added and mixed well and incubated on ice for 4 h. Centrifuging at 12000rpm for 15min at 4 deg.C, discarding supernatant, adding 1mL TBS to resuspend phage precipitate, filtering with 0.22 μm filter membrane, adding 20% sterilized glycerol, and storing at-20 deg.C to obtain nanometer antibody phage library.

Example 2 screening and identification of anti-carbofuran pesticide Nanobody

First, experiment method

1. BFNB-OVA was used as the coating antigen, 100. mu.l of each well of the ELISA plate was incubated overnight in a water bath at 37 ℃. The coating concentration gradient was 10, 5, 1, 0.2. mu.g/ml. After 12h, the plates were washed 2 times with PBST, 120. mu.l of 1% BSA was added to 10 and 1. mu.g/ml wells, and 120. mu.l of 1% fish collagen was added to 5 and 0.2. mu.g/ml wells, blocked for 3h, and dried at 37 ℃ until use.

2. 100 μ l of phage pool (about 10) was taken¹¹pfu), 2% BSA, OVA and KLH carrier proteins were added, respectively, and incubated at 37 ℃ for 1h to remove the antibody carried by the non-specific adsorption carrier. Then transferred to antigen-coated wells, shaken at room temperature for 1 hour, aspirated of unbound phage, and washed 5, 10, 15 times with PBST. Phage antibodies adsorbed in the wells of the plate were eluted with 100. mu.l of eluent (2, 1, 0.5, 0.1. mu.g/ml carbofuran). 10 μ l was removed for titer determination, and the remaining eluted product was subjected to helper phage rescue amplification and used for the next round of panning. A total of 4 rounds of panning were performed (see table 2). Phage monoclonals from the third and fourth rounds were randomly picked for indirect competition ELISA.

Table 2 panning policies table:

Recovery＝output phage/input phage

Enrichment＝after round/previous round

second, experimental results

The indirect competition ELISA results of the third and fourth rounds of phage monoclonals are shown in FIG. 1, and nine kinds of nanobodies with different sequences are obtained: nb309(393bp), Nb316(393bp), Nb328(375bp), Nb391(393bp), Nb393(372bp), Nb415(372bp), Nb438(372bp), Nb480(384bp), Nb489(366bp) antibodies. Through screening, the Nb316 has the best inhibition effect, and the amino acid sequence is shown as SEQ ID NO. 1; the nucleotide sequence is shown as SEQ ID NO. 2.

Example 3 preparation of carbofuran pesticide Nanobody and establishment of Indirect competitive ELISA method

First, experiment method

1. The Nb316-pComb3xss plasmid was extracted by means of an extraction kit and then introduced into competent E.coli BL21(DE3) by means of chemical transformation. Taking a single clone to carry out PCR identification and sequencing, and determining the insert as a target fragment. BL21(DE3) colonies containing nanobody target fragments were cultured to log-phase OD₆₀₀The value is 0.6-0.8, IPTG with the final concentration of 1mM is added, and the induction expression is carried out for 12h at 37 ℃. The next day, the cells were centrifuged. Then extracting periplasmic cavity protein by a sucrose osmotic pressure method, and recovering a soluble nano antibody Nb316 in the periplasmic cavity after one-step Ni column purification.

2. BFNB-OVA was used as the coating antigen, diluted to a working concentration of 1. mu.g/mL, added to a 96-well microplate, 100. mu.L per well, and incubated overnight at 37 ℃. The next day, washing the plate for 2 times, spin-drying the liquid in the pores, adding 120 μ L of confining liquid, incubating at 37 deg.C for 2h, spin-drying the liquid in the pores, pouring in an oven at 37 deg.C, oven-drying for 1h, and taking out for use. 50 μ L of the drug diluted in a gradient and 50 μ L of nanobody were added to each well in triplicate for each concentration. Incubating at 37 ℃ for 1h, washing the plate 5 times with a plate washing machine, spin-drying the liquid in the pores, adding 100 μ L of anti-HA-HRP antibody (20000 times dilution) into each pore, incubating at 37 ℃ for 40min, washing the plate 5 times with a plate washing machine, and spin-drying the liquid in the pores. Adding 100 μ L of TMB developing solution into each well, incubating at 37 deg.C for 10min, and adding 50μ L of 10% H₂SO₄The color reaction is stopped, and the enzyme-linked immunosorbent assay (A) is carried out by an enzyme-linked immunosorbent assay (ELIASA)_450nmAnd (4) light absorption value. The concentration of the carbofuran is taken as the abscissa, and the ratio of the light absorption values B/B₀For ordinate, the standard curve was fitted using a four parameter fitting module of origine 9.0.

Second, experimental results

The results show that the indirect competition ELISA standard curve (shown in figure 2), IC (integrated circuit) based on the nano antibody Nb2 for detecting carbofuran₅₀The linear range is 1.44-30.40 ng/ml, and the linear range is 7.27 +/-0.88 ng/ml.

Example 4 Indirect competitive ELISA for detection of various carbamate pesticides

First, experiment method

1. BFNB-OVA is used as a coating antigen, Nb316 is used as a detection antibody, and the specificity and sensitivity of the carbamate pesticide are evaluated by adopting an indirect competition ELISA method.

The method comprises the following specific steps: 50 μ L of the drug diluted in a gradient and 50 μ L of nanobody were added to each well in triplicate for each concentration. Incubating at 37 ℃ for 1h, washing the plate 5 times with a plate washing machine, spin-drying the liquid in the pores, adding 100 μ L of anti-HA-HRP antibody (20000 times dilution) into each pore, incubating at 37 ℃ for 40min, washing the plate 5 times with a plate washing machine, and spin-drying the liquid in the pores. Adding 100 μ L of TMB developing solution into each well, incubating at 37 deg.C for 10min, and adding 50 μ L of 10% H₂SO₄The color reaction is stopped, and the enzyme-linked immunosorbent assay (A) is carried out by an enzyme-linked immunosorbent assay (ELIASA)_450nmAnd (4) light absorption value. The concentration of the carbofuran is taken as the abscissa, and the ratio of the light absorption values B/B₀For ordinate, the standard curves were fitted using a four parameter fitting model from origin9.0 to obtain the respective ICs₅₀The value is obtained. The cross-reactivity rate of each drug with anti-carbofuran nanobody was calculated using the following formula:

CR(％)＝100×IC₅₀(carbofuran)/IC₅₀(carbofuran analogs).

2. The results show that the cross-reactivity rates of the nano antibody Nb316 with benfuracarb, fenobucarb, carbosulfan, 3-hydroxy carbofuran and isoprocarb are respectively 5.05%, 3.52%, 2.56%, 1.97% and 0.35%, and the cross-reactivity rate with the other 7 pesticides is lower than 0.1% (see Table 3).

TABLE 3 ic-ELISA method for detecting pesticide sensitivity and specificity of the Nanobody Nb 316:

example 5 thermal stability analysis of carbofuran pesticide nanobody

First, experiment method

1. The nano antibody Nb316 is diluted to the working concentration, divided into seven equal parts, and respectively placed in water baths at 20, 35, 50, 65, 80 and 95 ℃ for heating for 5 min. After the antibody is returned to room temperature, the binding capacity of the antibody and the antigen is determined by ic-ELISA, the binding capacity of the antibody without heating to the antigen is taken as 100%, and the stability of the nanobody at different temperatures is evaluated.

2. Diluting the nano antibody Nb316 to the working concentration, dividing into 7 equal parts, placing in a water bath kettle at 95 ℃, and heating for 10min, 20min, 30min, 40min, 50 min and 60min respectively. After the antibody was returned to room temperature, the binding ability of the antibody to the antigen was measured by the ic-ELISA of example 4, and the ability of the antibody to bind to the antigen without heating was taken as 100%, and the change in stability of the nanobody with time at high temperature was evaluated.

Second, experimental results

The results show that: the nano antibody Nb316 can still keep more than 100% of activity at 95 ℃ for 5min, so the nano antibody still has stronger binding capacity at high temperature (figure 3). The nanobody remained at about 100% binding activity at 95 ℃ for 0min to 60min (fig. 4).

Example 6 analysis of organic solvent resistance of carbofuran pesticide nanobody

First, experiment method

The nanobody Nb316 was diluted to the same working concentration using methanol and acetonitrile at different concentrations (10%, 20%, 40%, 60% and 80%) and the binding ability of the antibody to the antigen was measured by the ic-ELISA of example 4, and the antibody binding ability without the dilution with the organic solvent was taken as 100%, and the tolerance of the nanobody to different organic solvents and different concentrations of the same organic solvent was evaluated.

Second, experimental results

The results show that: the nanobody Nb316 still can maintain about 100% of the binding activity at a methanol concentration of 25%, and the binding activity still remains 50% at a methanol concentration of 50% (fig. 5). In the acetonitrile environment, the binding activity of the nano antibody cannot be reduced when the concentration of the acetonitrile is lower than 20%, and the nano antibody still has 50% binding activity when the concentration of the acetonitrile is 30% (figure 6). Therefore, the nano antibody has excellent organic solvent tolerance, and cannot be influenced by residual organic solvent in the pretreatment process of actual sample detection.

Sequence listing

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Claims

1. A nano antibody for specifically recognizing carbofuran is characterized in that the amino acid sequence of the nano antibody is shown as SEQ ID NO. 1.

2. A gene encoding the carbofuran-nanobody of claim 1, which specifically recognizes.

3. A recombinant vector comprising the gene of claim 2 linked thereto.

4. A recombinant cell carrying the recombinant vector of claim 3, or a cell capable of expressing the nanobody of claim 1.

5. Use of one or more of the nanobody of claim 1, the gene of claim 2, the recombinant vector of claim 3, or the recombinant cell of claim 4 in the preparation of a carbofuran detection kit.

6. The method for preparing nanobody of claim 1, comprising the steps of:

s1, connecting the coding gene of the nano antibody of claim 2 to an expression vector to obtain a recombinant vector;

and S4, separating and purifying to obtain the nano antibody.

7. A carbofuran assay for non-therapeutic or diagnostic purposes, using the nanobody of claim 1.

8. The method of claim 7, wherein the detection is performed by indirect ELISA, and comprises using the carbofuran hapten of formula (I) coupled with a carrier protein as a coating antigen, and using the nanobody of claim 1 as a detection antibody

9. A kit for detecting carbofuran, which comprises the nanobody of claim 1.

10. The kit of claim 9, wherein the detection is performed based on an indirect ELISA method, and further comprises a carbofuran complete antigen obtained by coupling carbofuran hapten and carrier protein of formula (I) as a coating antigen, and the nano-antibody of claim 1 is used as a detection antibody